Atomic Force Microscopy of DNA and DNA-Protein Interactions.

Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometer resolution. For biological applications, one of its key advantages is its ability to visualize the substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine the secondary and tertiary structure of surface-bound DNA, and its interactions with proteins.

Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization.

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear enzyme that plays important roles in DNA repair, chromatin organization and transcription regulation. Although binding and activation of PARP1 by DNA damage sites has been extensively studied, little is known about how PARP1 binds to long stretches of undamaged DNA and how it could shape chromatin architecture. Here, using single-molecule techniques, we show that PARP1 binds and condenses undamaged, kilobase-length DNA subject to sub-piconewton mechanical forces. Stepwise decondensation at high force and DNA braiding experiments show that the condensation activity is due to the stabilization of DNA loops by PARP1. PARP inhibitors do not affect the level of condensation of undamaged DNA but act to block condensation reversal for damaged DNA in the presence of NAD+ Our findings suggest a mechanism for PARP1 in the organization of chromatin structure.

Atomic force microscopy-A tool for structural and translational DNA research.

Atomic force microscopy (AFM) is a powerful imaging technique that allows for structural characterization of single biomolecules with nanoscale resolution. AFM has a unique capability to image biological molecules in their native states under physiological conditions without the need for labeling or averaging. DNA has been extensively imaged with AFM from early single-molecule studies of conformational diversity in plasmids, to recent examinations of intramolecular variation between groove depths within an individual DNA molecule. The ability to image dynamic biological interactions in situ has also allowed for the interaction of various proteins and therapeutic ligands with DNA to be evaluated-providing insights into structural assembly, flexibility, and movement. This review provides an overview of how innovation and optimization in AFM imaging have advanced our understanding of DNA structure, mechanics, and interactions. These include studies of the secondary and tertiary structure of DNA, including how these are affected by its interactions with proteins. The broader role of AFM as a tool in translational cancer research is also explored through its use in imaging DNA with key chemotherapeutic ligands, including those currently employed in clinical practice.

PEGylated surfaces for the study of DNA-protein interactions by atomic force microscopy.

DNA-protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA-protein interactions at nanometre resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working in biologically relevant protein concentrations, where proteins may be present in large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface for AFM studies. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, we show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113/PLL1000-2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. We show - through the use of biotinylated DNA and streptavidin - that this selective adsorption extends to DNA-protein complexes and that DNA-bound streptavidin can be unambiguously distinguished in spite of an excess of unbound streptavidin in solution. Finally, we apply this to the nuclear enzyme PARP1, resolving the binding of individual PARP1 molecules to DNA by in-liquid AFM.